HPV E6, E7 mRNA ASSAY AND METHODS OF USE THEREOF

ABSTRACT

Provided is an HPV E6, E7 mRNA assay, referenced herein as the “In Cell HPV Assay,” that is capable of sensitive and specific detection of normal cervical cells undergoing malignant transformation as well as abnormal cervical cells with pre-malignant or malignant lesions. The In Cell HPV Assay identifies HPV E6, E7 mRNA via in situ hybridization with oligonucleotides specific for HPV E6, E7 mRNA and quantitates the HPV E6, E7 mRNA via flow cytometry. The In Cell HPV Assay can be carried out in less than three hours directly from liquid-based cervical (“LBC”) cytology specimens. The In Cell HPV Assay provides an efficient and highly sensitive alternative to the Pap smear for determining abnormal cervical cytology.

TECHNICAL FIELD

The invention relates generally to an HPV mRNA assay for the detectionof HPV-related disorders such as cervical cancer and cervical lesions.More specifically, the invention relates to an assay that identifies andquantitates HPV E6, E7 mRNA in cervical cells. The HPV E6, E7 mRNAAssay, referenced herein as the “In Cell HPV Assay,” is capable ofsensitive and specific detection of cancerous and pre-cancerous abnormalcells as well as normal cells undergoing malignant transformation.

BACKGROUND OF THE INVENTION

Cervical cancer affects approximately 13,000 women per year in theUnited States and more than 400,000 women worldwide. Ninety percent ofcervical cancers contain the high risk HPV DNA strains 16 and 18(“HPV-16” and “HPV-18,” respectively). By contrast, low risk HPVs, suchas HPV-6 and HPV-11, rarely develop into cancer. The presence of lowrisk and high risk HPVs are identified through the use of the polymerasechain reaction (“PCR”) or the Hybrid Capture® II HPV Test (“HC II HPVTest”; Digene Corp., Gaithersburg, Md.)

The Papanicolaou (“Pap”) smear assesses a patient's risk for cervicalcancer by testing for the presence of squamous intraepithelial lesions(“SILs”) on the cervix. The Pap smear has been the standard of care inthe U.S. for over 50 years, resulting in a 74% decline in deaths due tocervical cancer. The Pap smear, however, is not without itsshortcomings; in particular, errors in cervical sampling andinterpretation contribute to a Pap smear sensitivity of only 58%.Hakama, Screening for Cervical Cancer, CANCER TREND RES. 86:41-49(1996); Nanda et al., Accuracy of the Papanicolaou Test in Screening forand Follow-up of Cervical Cytologic Abnormalities, ANN. INTERN. MED.132:810-819 (2000). In 1996, the Food and Drug Administration approvedthe ThinPrep® Pap Test (Cytec Corp., Marlborough, Mass.) as analternative to the conventional Pap smear for the screening of SILs. TheThinPrep Pap Test screens for SILs using liquid based cytology (“LBC”)with automated monolayer slide production. The use of LBC has resultedin an increase in adequate specimens and the detection of SILs; however,LBC samples have a sensitivity of only 80%. Corkill et al., SpecimenAdequacy of the ThinPrep Sample Preparations in a Direct-to-Vial Study,ACTA CYTOL. 41:39-44 (1997); Wilbur et al., Clinical Trials of theCYTORICH Specimen-Preparation Device for Cervical Cytology, ACTA CYTOL.41:24-29 (1997).

While the life cycle of HPV would indicate that women with high riskHPVs will develop either low grade SILs (“LGSILs,” i.e., SILs from anearly pre-malignant lesion) or high grade SILs (“HGSILs,” i.e., SILsfrom an advanced pre-malignant lesion) and progress to cancer whilewomen with low risk HPVs will not; in reality, only a minority of womeninfected with high risk HPVs and exhibiting either LGSILs or HGSILs willprogress to cancer. The following table shows the rate of regression andprogression of women diagnosed via Pap smear or LBC with ASCUS (abnormalsquamous cells of undetermined significance), LGSILs, and HGSILs (fromMelnikow et al., Natural History of Cervical Squamous IntraepithelialLesions: A Meta-Analysis, J. OBSTET. GYNECOL. 92:727-735 (1998); seealso, Woodman et al., Natural History of Cervical Human PapillomavirusInfection in Young Women: A Longitudinal Cohort Study, LANCET357:1831-1836 (2001)):

PROGRESSION TO PROGRESSION TO REGRESSION TO HIGHER GRADE OVER INVASIVECANCER CYTOLOGY NORMAL 24 MONTHS OVER 24 MONTHS ASCUS 68%  7% 0.25%LGSIL 47% 21% 0.15% HGSIL 35% 23% 1.44%

As the data in the foregoing table demonstrates, the majority of womendiagnosed with ASCUS, LGSILs, and HGSILs do not progress to cancer;accordingly, the traditional Pap smear and LBC test, both of whichmerely identify the presence abnormal SILs are not effective tests todistinguish benign lesions from lesions with malignant potential.Similarly, because the majority of women diagnosed with ASCUS, LGSILs,and HGSILs are usually infected with a high risk HPV, it follows thatthe identification of infection with a high risk HPV is also not abiologically relevant indicator for cervical cancer detection. There istherefore, a need in the art for a more highly sensitive method by whichto screen for cervical cancer.

SUMMARY OF THE INVENTION

The present invention overcomes the need in the aforementioned need inthe art by providing assays and method for screening cervical cancer byscreening for, detecting, and quantitating intracellular HPV E6, E7 mRNAin cervical cells. To the best of the inventor's knowledge, no such HPVE6, E7 mRNA assay has been previously published or disclosed.

In a first aspect of the invention, there is provided an assay fordetermining malignant transformation of cervical cells comprising thesteps of: (a) obtaining cervical cells from an individual; (b) preparingthe cervical cells as a liquid-based cytology (LBC) specimen; and (c)hybridizing the LBC specimen using oligonucleotides specific for HPV E6,E7 mRNA, wherein expression of HPV E6, E7 mRNA indicates malignanttransformation of the cervical cells. Within this aspect of theinvention, the HPV E6, E7 mRNA may be quantitated via flow cytometry.

In a second aspect of the invention, there is provided a method ofscreening a patient for malignant transformation of cervical cellscomprising identifying expression of HPV E6, E7 mRNA in the cervicalcells. In a preferred embodiment, the HPV E6, E7 mRNA is identifiedusing in situ hybridization with oligonucleotides specific for HPV E6,E7 mRNA and the HPV E6, E7 mRNA is quantitated using flow cytometry.

Generally malignant transformation of the cervical cells is indicated byexpression of 2 to 1000 copies of HPV E6, E7 mRNA per cell, withexpression of 5 to 750 copies of HPV E6, E7 mRNA per cell preferred,expression of 10 to 500 copies of HPV E6, E7 mRNA per cell morepreferred, and expression of 200 or more copies of HPV E6, E7 mRNA percell most preferred.

The cervical cells that may be used in both the assay and the methoddescribed above are selected from the group consisting of normal cells,abnormal squamous cells of undetermined significance (ASCUS), cells withlow grade squamous intraepithelial lesions (LGSILs), and cells with highgrade intraepithelial lesions (HGSILs).

In a third aspect of the invention, there is provided a method ofscreening a patient with abnormal cervical cells for pre-malignantcervical cells, comprising quantitating the expression of HPV E6, E7mRNA in the cervical cells, wherein cells expressing 200 or more copiesof HPV E6, E7 mRNA per cell indicate cells with malignant potential andcells expressing less than 200 copies of HPV E6, E7 mRNA indicate benigncells. The abnormal cervical cells are selected from the groupconsisting of abnormal squamous cells of undetermined significance(ASCUS), cells with low grade squamous intraepithelial lesions (LGSILs),and cells with high grade intraepithelial lesions (HGSILs). In apreferred embodiment, the HPV E6, E7 mRNA is identified using in situhybridization with oligonucleotides specific for HPV E6, E7 mRNA and theHPV E6, E7 mRNA is quantitated using flow cytometry.

In a fourth aspect of the invention, there is provided an assay foridentifying malignantly transformed cervical cell types comprising thesteps of: (a) obtaining cervical cells from an individual; (b) preparingthe cervical cells as a liquid-based cytology (LBC) specimen; (c)staining the cervical cells with a combination of CD16 and CAM 5.2; and(d) hybridizing the LBC specimen using oligonucleotides specific for HPVE6, E7 mRNA, wherein neutrophils are identified by CD16⁺ staining,columnar endocervical cells are identified by CD16⁻, CAM 5.2⁺ staining,and squamous ectocervical cells are identified by CD16⁻, CAM 5.2⁻staining, and expression of HPV E6, E7 mRNA indicates malignanttransformation of the cervical cells. Within this aspect of theinvention, the expression of HPV E6, E7 mRNA per cell may be quantitatedvia flow cytometry, with malignant transformation of the cervical cellsindicated by expression of 2 to 1000 copies of HPV E6, E7 mRNA per cell,with expression of 5 to 750 copies of HPV E6, E7 mRNA per cellpreferred, expression of 10 to 500 copies of HPV E6, E7 mRNA per cellmore preferred, and expression of 200 or more copies of HPV E6, E7 mRNAper cell most preferred. Malignantly transformed columnar endocervicalcells indicate a pre-adenocarcinoma or adenocarcinoma cervical conditionand malignantly transformed squamous ectocervical cells indicatepre-squamous cell carcinoma or squamous cell carcinoma cervicalcondition.

Additional aspects, advantages and features of the invention will be setforth, in part, in the description that follows, and, in part, willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with the color drawing will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a graph showing the relationship between the mean fluorescenceintensity (“MFI”) of HPV E6, E7 mRNA quantitated by the In Cell HPVAssay using flow cytometry for quantification versus copies of HPV E6mRNA quantitated by real-time reverse transcriptase PCR (“real-timeRT-PCR”) in HPV⁻ normal ectocervical cells and HPV⁺ SiHa and HeLa cells.

FIG. 2 is a graph showing the linear relationship between serialdilutions of a mixed population of HPV⁺ HeLa cells in HPV⁻ normalectocervical cells versus the percentage of cells in the mixedpopulation expressing HPV E6, E7 mRNA quantitated by the In Cell HPVAssay using flow cytometry.

FIGS. 3A-3E collectively show the detection and morphology of abnormaland normal cells stained on slides (scale bar 20 μm). FIG. 3A shows themorphology of abnormal cells expressing HPV E6, E7 mRNA and FIG. 3Bshows the morphology of normal squamous epithelial cells. FIGS. 3C-3Eshow that the abnormal cells have a characteristically high nuclear tocytoplasmic ratio (arrows) whereas the normal cells exhibit a lownuclear to cytoplasmic ratio (arrowheads).

FIGS. 4A-4I show flow cytometry histograms of normal and abnormalcervical cells gated by light scatter, stained with a combination ofCD16⁻ and CAM 5.2⁻ to identify ectocervical cells, and analyzed for HPVE6, E7 mRNA expression using the In Cell HPV Assay of the presentinvention. FIG. 4A shows the analysis of cervical cells by forward andside scatter (first box) with a sample (first inner box) selected forstaining for ectocervical cells (second box) and with a selected sample(second inner box) of ectocervical cells analyzed for HPV E6, E7 mRNAusing the In Cell HPV Assay with 100 copies per cell used as the cut-offpoint for determining the percentage of cells in a sample expressing HPVE6, E7 mRNA. FIGS. 4B-4E show the assay applied to four women withabnormal cytology (HGSIL) and FIGS. 4F-4I show the assay applied to fourwomen with normal cytology (WNL). The percentage of cells expressingmore than 100 copies of HPV E6, E7 mRNA per cell in each sample isindicated in the upper right hand corner of the boxes.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Nomenclature

Before describing detailed embodiments of the invention, it will beuseful to set forth definitions that are used in describing theinvention. The following definitions are used in describing theinvention. The terminology used in the definitions is for the purpose ofdescribing the particular embodiments of the invention and is notintended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise.

The term “In Cell HPV Assay” refers to the simultaneousimmunofluorescence and ultrasensitive fluorescence in situ hybridizationassay described in Examples 1 and 2. The In Cell HPV Assay is capable ofidentifying cancerous and precancerous SILs by detecting the presence ofvery low copies of HPV E6, E7 mRNA in the SILs. Where appropriate, theHPV E6, E7 mRNA detected by the In Cell HPV Assay may be quantitatedusing flow cytometry; thus, as used herein, the term “In Cell HPV Assay”is used generally to refer to the identification and where appropriate,the quantitation of the HPV E6, E7 mRNA.

The term “Bethesda System” refers to the currently used classificationsystem for Pap smear and ThinPrep Pap test analysis. The Bethesda Systemis described at www.cytopathology.org/NIH and includes theclassifications WNL, ASCUS, LGSIL, and HGSIL already referenced hereinas well as the term “squamous cell carcinoma” for malignantlytransformed cervical cells. The Bethesda System classifications are alsoreferenced herein as “cytologies.”

As used herein, the term “cervical cells” refers generally to thesquamous epithelial cells that line the surface of the cervix. The termis used generally to include normal cervical cells as well the abnormalcervical cells, i.e., ASCUS, LGSILs, HGSILs, and squamous cellcarcinomas.

The term “squamous intraepithelial lesions” or “SILs” refers to lesionsthat form on the cervical squamous cells and includes both the low grade(LGSILs) and the high grade (HGSIL) lesions.

The term “adenocarcinoma” and “squamous cell carcinoma” are usedconventionally to refer to malignant epithelial cell tumors of theglands (adenocarcinoma) and malignant tumors of the squamous epithelium(squamous cell carcinoma). The terms “pre-adenocarcinoma” and“pre-squamous cell carcinoma” refer to lesions that have not yet beenmalignantly transformed. As used herein, the terms adenocarcinomas andpre-adenocarcinomas refer to tumors and lesions of the glandularepithelium of the cervix and the terms squamous cell carcinomas andpre-squamous cell carcinomas refer to tumors and lesions of the squamousepithelial cells of the cervix.

The term “nucleic acid analyte” refers generally to all nucleic acids,such as all DNAs and RNAs, and segments thereof. Within the context ofthe present invention, the term includes DNA, RNA, and mRNA.

As used herein, the term “target amplification” refers toenzyme-mediated procedures that are capable of producing billions ofcopies of nucleic acid target. Examples of enzyme-mediated targetamplification procedures known in the art include PCR, nucleicacid-sequence-based amplification (“NASBA”), transcription-mediatedamplification (“TMA”), strand displacement amplification (“SDA”), andligase chain reaction (“LCR”).

The most widely used target amplification procedure is PCR, firstdescribed for the amplification of DNA by Mullins et al. in U.S. Pat.No. 4,683,195 and Mullis in U.S. Pat. No. 4,683,202. The PCR procedureis well known to those of ordinary skill in the art. Where the startingmaterial for the PCR reaction is RNA, complementary DNA (“cDNA”) is madefrom RNA via reverse transcription. A PCR used to amplify RNA productsis referred to as reverse transcriptase PCR or “RT-PCR.”

As used herein, the term “type specific PCR” refers to the use of PCR toidentify high risk and low risk HPV DNA.

Real-time PCR and Real-time RT-PCR refers to the detection of PCRproducts via a fluorescent signal generated by the coupling of afluorogenic dye molecule and a quencher moiety to the same or differentoligonucleotide substrates. Examples of commonly used probes used inreal-time PCR and real-time RT-PCR include the following probes: Taqman®probes, Molecular Beacons probes, Scorpions® probes, and SYBR® Greenprobes. Briefly, Taqman probes, Molecular Beacons, and Scorpion probeseach have a fluorescent reporter dye (also called a “fluor”) attached tothe 5′ end of the probes and a quencher moiety coupled to the 3′ end ofthe probes. In the unhybridized state, the proximity of the fluor andthe quench molecules prevents the detection of fluorescent signal fromthe probe; during PCR, when the polymerase replicates a template onwhich a probe is bound, the 5′-nuclease activity of the polymerasecleaves the probe thus, increasing fluorescence with each replicationcycle. SYBR Green probes binds double-stranded DNA and upon excitationemit light; thus as PCR product accumulates, fluorescence increases.

Within the context of the present invention, real-time RT-PCR is used toidentify oncogenic mRNA in abnormal cervical samples, such as forexample, the E6 mRNA found in HPV-16 and HPV-18. Real-time RT-PCR isused in Examples 6 and 7 with a Taqman probe. Taqman probes are designedto hybridize to an internal region of a PCR product. In the unhybridizedstate, the proximity of the fluor and the quench molecules prevents thedetection of fluorescent signal from the probe; during PCR, when thepolymerase replicates a template on which a Taqman probe is bound, the5′-nuclease activity of the polymerase cleaves the probe thus,increasing fluorescence with each replication cycle.

The term “signal amplification” refers to techniques that are capable ofdetecting and quantitating the amount of nucleic acid analyte in asample by amplifying the signal of a target in direct proportion to theamount of target. Because the amount of target is not altered, signalamplification procedures are useful for quantitating the amount ofnucleic acid analyte in as sample. Examples of signal amplificationprocedures known in the art include branched DNA (“bDNA”) signalamplification (Bayer Healthcare LLC, Tarrytown, N.Y.), Hybrid Capture(“HC”; Digene Corp., Gaithersburg, Md.), and Tyramide SignalAmplification (“TSA,” Perkin Elmer, Inc., Wellesley, Mass.).

Within the context of the present invention, the HC II HPV Test is asignal amplification assay used to detect the presence of HPV DNA inclinical samples. The HC II HPV Test includes the following steps:clinical specimens are combined with a base solution that disrupts thevirus and releases target DNA; target DNA combines with specific RNAprobes creating RNA:DNA hybrids; multiple RNA hybrids are captured ontoa solid phase coated with universal capture antibodies specific forRNA:DNA hybrids; captured RNA:DNA hybrids are detected with multipleantibodies conjugated to alkaline phosphatase (“AP”; amplification mayreach 3000 fold); and the bounds alkaline phosphatase is detected with achemiluminescent dioxetane substrate. Upon cleavage by AP, the substrateproduces light that may be measured on a luminometer in relative lightunits (“RLUs”). The HC II HPV Test is cap able of detecting 13 high-riskHPV types: HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45,HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, and HPV-68.

Identifying and Quantitating HPV E6, E7 mRNA

The present invention provides an HPV E6, E7 mRNA assay (the In Cell HPVAssay) that identifies pre-cancerous and cancerous cervical cells bydetecting the presence of HPV E6, E7 mRNA in the cells. Because HPV E6,E7 mRNA is a highly sensitive biomarker for the detection of malignanttransformation of cervical cells, the In Cell HPV Assay described hereinis capable of detecting malignant transformation of cervical cells thatexhibit normal cytology in addition to cells exhibiting abnormalcytology, e.g., ASCUS, LGSILs, and HGSILs (Examples 6 and 7).

The In Cell HPV Assay consists of an in situ hybridization assayperformed with HPV E6, E7 mRNA oligonucleotide probes, and whereappropriate, flow cytometry to quantitate the HPV E6, E7 mRNA. Examples1 and 2 describe two different in situ hybridization methodologies tocarry out the In Cell HPV Assay and Example 3 describes the use of flowcytometry to quantitate the HPV E6, E7 mRNA.

Based upon the foregoing principles, in a first embodiment of thepresent invention, there is provided an assay for determining cytologyof cervical cells comprising the steps of: (a) obtaining cervical cellsfrom an individual; (b) preparing the cervical cells as an LBC specimen;and (c) hybridizing the LBC specimen using oligonucleotides specific forHPV E6, E7 mRNA, wherein expression of HPV E6, E7 mRNA indicatesmalignant transformation of the cervical cells. Within this firstembodiment of the present invention, the HPV E6, E7 mRNA may bequantitated via flow cytometry.

In a second embodiment of the present invention, there is provided amethod of screening a patient for malignant transformation of cervicalcells comprising identifying expression of HPV E6, E7 mRNA in thecervical cells. Within this second embodiment, the HPV E6, E7 mRNA isidentified using in situ hybridization and the HPV E6, E7 mRNA isquantitated using flow cytometry.

In both the assay and method of the first and second embodiments of thepresent invention, the cervical cells are selected from the groupconsisting of normal cells, abnormal squamous cells of undeterminedsignificance (ASCUS), cells with low grade squamous intraepitheliallesions (LGSILs), and cells with high grade intraepithelial lesions(HGSILs). As mentioned above, a significant advantage of the In Cell HPVAssay over the other available methods of detecting cervical cellcytology is that the In Cell HPV Assay, through the detection of HPV E6,E7 mRNA expression, is capable of identifying early malignanttransformation in otherwise normal appearing cells. None of thepresently available methods of cervical cell screening are capable ofsuch sensitivity and specificity. The ability of the In Cell HPV Assayto detect HPV E6, E7 mRNA in cells with normal cytology is described inExamples 6 and 7. In Example 6, 3 of 41 high risk women with normalcervical cytology tested positive for HPV E6, E7 mRNA using the In CellHPV Assay and in Example 7, 10 of 109 low risk women tested positive forHPV E6, E7 mRNA; thus, indicating malignant transformation in the“normal” cells of these women.

As noted above, the diagnostic results of the In Cell HPV Assay wereconfirmed in Examples 6 and 7 using real-time RT-PCR and in Example 7using both real-time RT-PCR and the HC II HPV Test. As previously noted,and as reiterated in Example 7, the In Cell HPV Assay of the presentinvention is superior over the HC II HPV Test for the detection ofcervical cancer because the In Cell HPV Assay is capable of detectingmalignantly transformed cells at a very early stage, while the HC II HPVTest only detects high risk HPV DNA, i.e., HPV-16 and HPV-18, both ofwhich are not indicators of malignant transformation of cells. The InCell HPV Assay is also superior over the use of real-time RT-PCR for thedetection of cervical cancer because unlike real time PCR, which is atarget amplification procedure that can only identify amplification of apopulation of cells, the In Cell HPV Assay is a highly specificprocedure that is capable of identifying amplification of an individualcell. In other words, comparing the amplification of one million copiesof HPV E6, E7 mRNA derived by real time RT-PCR versus one million copiesof HPV E6, E7 mRNA derived by the In Cell HPV Assay of the presentinvention, the real-time RT-PCR will only be able to discern thatone-million copies of the gene have been produced; by contrast, the InCell HPV Assay is capable of discerning exactly how many cells haveproduced the one million copies. Thus, under real-time RT-PCR, theordinary practitioner will be unable to identify if one millionindividual cells produced the one million copies as occurs in a normalindividual or if one thousand cells produced one thousand cells each asoccurs in cancer. The In Cell HPV Assay of the present inventionovercomes this deficiency in real-time RT-PCR by identifying, via flowcytometric quantitation, the precise number of copies of HPV E6, E7 mRNAthat are produced per cell. In this way, in Examples 6 and 7, thereal-time RT-PCR was used only to verify the presence of HPV E6, E7mRNA.

In both the first and second embodiments of the present invention, themalignant transformation of the cervical cells is indicated byexpression of 2 to 1000 copies of HPV E6, E7 mRNA per cell, withexpression of 5 to 750 copies of HPV E6, E7 mRNA per cell preferred,expression of 10 to 500 copies of HPV E6, E7 mRNA per cell morepreferred, and expression of 200 or more copies of HPV E6, E7 mRNA percell per cell most preferred. While the theoretical sensitivity of theflow cytometric analysis in Example 3 was found to be 10 to 20 copies ofHPV E6, E7 mRNA, further calibration of the flow cytometric assay shouldtheoretically reduce the sensitivity of the flow cytometric analysis toas low as one to two copies of HPV E6, E7 mRNA and as high as 1000copies of HPV E6, E7 mRNA per cell.

In a third embodiment of the present invention, there is provided amethod of screening a patient with abnormal cervical cells forpre-malignant cervical cells, comprising quantitating the expression ofHPV E6, E7 mRNA in the cervical cells, wherein cells expressing 200 ormore copies of HPV E6, E7 mRNA per cell indicate cells with malignantpotential and cells expressing less than 200 copies of HPV E6, E7 mRNAindicate benign cells. The abnormal cells that may be tested in thisthird embodiment may be selected from the group consisting of abnormalsquamous cells of undetermined significance (ASCUS), cells with lowgrade squamous intraepithelial lesions (LGSILs), and cells with highgrade intraepithelial lesions (HGSILs). Similar to the previouslydiscussed embodiments, the HPV E6, E7 mRNA is identified via in situhybridization and quantitated via flow cytometry.

Where desired, the specific morphology of the cervical cells may bedetermined by the procedure set forth in Example 5 and depicted in FIG.3. Specifically, by staining cervical cells with a combination of CD 16(a neutrophil marker) and CAM 5.2 (an antibody expressed in endocervicalcells but not ectocervical cells), it is possible to identify thecytology of the cells expressing the HPV E6, E7 mRNA. In a similar vein,the antibody staining formula of Example 5 may also be used to study aspecific class of cells, such as the expression levels of HPV E6, E7mRNA in squamous ectocervical cells from a specific high risk or lowrisk cohort (Example 6).

Based upon the principles exemplified in Examples 5 and 6, the fourthembodiment of the present invention provides an assay thatsimultaneously phenotypes malignantly transformed cells. This assay, foridentifying malignantly transformed cervical cell types includes thefollowing steps: (a) obtaining cervical cells from an individual; (b)preparing the cervical cells as a liquid-based cytology (LBC) specimen;(c) staining the cervical cells with a combination of CD16 and CAM 5.2;and (d) hybridizing the LBC specimen using oligonucleotides specific forHPV E6, E7 mRNA, wherein neutrophils are identified by CD16⁺ staining,columnar endocervical cells are identified by CD16⁻, CAM 5.2⁺ staining,and squamous ectocervical cells are identified by CD16⁻, CAM 5.2⁻staining, and expression of HPV E6, E7 mRNA indicates malignanttransformation of the cervical cells. Malignantly transformed columnarendocervical cells indicate a pre-adenocarcinoma or adenocarcinomacervical condition and malignantly transformed squamous ectocervicalcells indicate pre-squamous cell carcinoma or squamous cell carcinomacervical condition. Like the first and second embodiments describedabove, expression of HPV E6, E7 mRNA per cell may be quantitated viaflow cytometry, with malignant transformation of the cervical cellsindicated by expression of 2 to 1000 copies of HPV E6, E7 mRNA per cell,with expression of 5 to 750 copies of HPV E6, E7 mRNA per cellpreferred, expression of 10 to 500 copies of HPV E6, E7 mRNA per cellmore preferred, and expression of 200 or more copies of HPV E6, E7 mRNAper cell most preferred.

Utility:

The In Cell HPV Assay described herein is useful as a highly sensitiveand specific screening method for the detection of pre-cancerous andcancerous cervical cells, which may be performed in less than threehours. The In Cell HPV Assay is premised on the identification andquantitation of HPV E6, E7 mRNA, where the presence of elevated levelsof HPV E6, E7 mRNA in cervical cells indicates the malignanttransformation of the cells.

The In Cell HPV Assay is superior over presently available methods forcervical cell screening in that it can detect malignant transformationin cells exhibiting a normal cytology. Because of its remarkablesensitivity and specificity, the In Cell HPV Assay has utility as analternative and superior primary cervical cell screening method over thetraditional Pap smear or the ThinPrep Pap test, both of which merelyidentify abnormal cervical cells (i.e., ASCUS, LGSILs, and HGSILs), andPCR and the HC II HPV Test, both of which merely identify high risk fromlow risk HPV. As previously explained, neither cervical cytology nor theidentification of low risk or high risk HPVs are reliable markers fordetermining cervical cell malignancy. For those situations where PCR orthe HC II HPV Test is used, the In Cell HPV Assay finds utility as asecondary marker for patients that are HPV DNA positive. In other words,upon primary screening of HPV DNA via PCR or HC II HPV, the In Cell HPVAssay may be used to determine if the SILs exhibiting high risk HPV DNAhave been malignantly transformed.

In addition to the foregoing, the In Cell HPV Assay also finds utilityas a highly desirable alternative cervical cell screening method in thatit is capable of being performed in less than three hours withestablished experimental protocols. The foregoing demonstrates that theIn Cell HPV Assay is not only more accurate than any presently usedmethods to detect malignant transformation of cervical cells, but it isalso more time and cost effective.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All patents, publications, and other published documents mentioned orreferred to, both supra and infra, are incorporated by reference intheir entireties.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions of the invention. Efforts have been madeto ensure accuracy with respect to numbers (e.g., amounts, temperature,etc.) but some experimental error and deviations should, of course, beallowed for. Unless indicated otherwise, parts are parts by weight,temperature is degrees centigrade and pressure is at or nearatmospheric. All components were obtained commercially unless otherwiseindicated.

EXPERIMENTAL

Unless otherwise indicated, all formulations described herein wereperformed with commercially available products. The following protocolswere used in the Examples.

Subjects: Women undergoing routine cervical cytology at multiple siteswere enrolled in a study following informed consent. Cervical cytologyspecimens were collected using a cytobrush and preserved using SurePath™(Tripath Imaging, Burlington, N.C.) liquid-based cytology fixative. Thesmears were classified using The Bethesda System.

Cell Lines: HeLa cells were obtained from the American Type CultureCollection (Manassas, Va.) and grown according to instructions. Normalhuman ectocervical cells (Clonetics, Inc., now Cambrex, Inc., EastRutherford, N.J.) were grown in media supplied by the company.

Real-Time RT-PCR and HC II HPV Test: For target amplification ofoncogenic mRNA, real-time RT-PCR was performed using Taqman® probes(Applied Biosystems, Foster City, Calif.). For signal amplification, theHC II HPV Test was performed according to the manufacture's instructionsusing a high risk panel of probes.

Cytometry: Flow cytometry was performed using three-color analysis on aFACScan (BDIS, San Jose, Calif.) cytometer. Analysis was restricted toectocervical cells defined by light scatter characteristics and the lackof expression of the low molecular weight cytokeratin CAM 5.2.Polymorphonuclear neutrophils (“PMN”) were excluded from the analysis bygating on cells positive for the PMN marker CD16.

Image Analysis: Inage analysis was performed using an Olympus® laserconfocal microscope. Cells were included in the analysis if theyexhibited green fluorescence (fluorescein, HPV E6, E7 mRNA+) and bluefluorescence (DAPI, all cells) but lacked red fluorescence (PMNs,endocervical cells).

Statistical Analysis: Statistical analyses were performed using either at-test or a Mann-Whitney Rank Sum test. P values <0.05 were consideredstatistically significant.

Example 1 Simultaneous Immunofluorescence and UltrasensitiveFluorescence In Situ Hybridization to Detect HPV E6, E7 (“in Cell HPVAssay”)

A 1 mL aliquot was removed from the liquid-based cervical cytologyspecimen isolated from the subjects. The cells were pelleted bycentrifugation at 400×g and washed once in phosphate buffered saline(“PBS”), pH 7.4. Cells were resuspended in 100 μL of PBS, pH 7.4 andstained with a 1:10 dilution of phycoerythrin (“PE”)-conjugated anti-CAM5.2 and PE/cy5-conjugated anti-CD16 (BDPharmingen, San Diego, Calif.).The cells were then incubated at 4° C. for 20 minutes in the dark.Following incubation, the cells were fixed, permeabilized, washed oncein PBS, pH 7.4, pelleted by centrifugation at 400×g, washed again in2×SSC, and pelleted by centrifugation. The cells were then resuspendedin a hybridization mix consisting of 5×SSC, 30% formamide, and 100 μg/mLsheared salmon sperm DNA (“ssDNA”) and a cocktail of 5′- and3′-fluorescein labeled HPV E6, E7 mRNA oligonucleotide probes, which maybe obtained from Invirion, Inc., Frankfort, Mich., under the name “HPVOncoTect,” or may be prepared by techniques for the preparation ofoligonucleotides known in the art. See e.g., Caruthers et al., ChemicalSynthesis of Deoxyoligonucleotides by the Phosphoramidite Method,METHODS ENZYMOL 154:287-313 (1987). Hybridization was performed at 37°C. for 30 minutes and was followed by a 5 minute wash in 2×SSC, 0.1%Triton X-100, and a 15 minute wash in 0.1% SSC, 0.1% Triton X-100.

Cells prepared as such were resuspended in PBS, pH 7.4 with 2% fetalcalf serum for flow cytometric analysis.

Example 2 The Slide-Based in Cell HPV Assay

100 μL of cells (1×10⁶ cells/mL) were transferred from the liquid basedcervical cytology specimen isolated from the subjects and put into acytocentrifuge or liquid-based slide system. The slides were centrifugedat 800×g for 2 minutes at room temperature. Following a wash in 1×PBS,pH 7.4, the slides were incubated in 1× PermiFlow (Invirion, Inc.,Frankfort, Mich.) fixation/permeabilization reagent in a Coplin jar atroom temperature for 1 hour. The slides were then washed once in PBS andonce in 2×SSC. The cells were hybridized to a cocktail of the HPVOncoTect probes a hybridization oven at 37° C. for 30-120 minutes. Theslides were then washed for 5 minutes in a Coplin jar containing 50 mLpre-heated 2×SSC, 0.1% Triton X-100 and incubated for 15 minutes in aCoplin jar containing 50 mL pre-heated 0.1×SSC, 0.1% Triton X-100.Following a brief rinse in PBS, pH 7.4, the slides were coverslippedusing Fluorsave mounting medium (CalBioChem, San Diego, Calif.).

Example 3 Validation of the in Cell HPV Assay

To validate the effectiveness of the In Cell HPV Assay, commerciallyavailable normal ectocervical cells, i.e., HPV⁻ ectocervical cells, andHPV⁺ SiHa and HeLa cells were grown in culture and split into 20 samplesand analyzed for HPV E6, E7 mRNA by the In Cell HPV Assay with flowcytometry and for HPV E6 mRNA by real-time RT-PCR with a detection limitof 10 copies per cell. FIG. 1 shows the relationship between the MFI ofHPV E6, E7 mRNA and copies of HPV E6 mRNA in each of the cellpopulations. Linear regression analysis of the results of FIG. 1resulted in a correlation of 0.88 with a p value of 0.001. Based uponthe results of this experiment, the theoretical sensitivity of the InCell HPV Assay was calculated to be 10 to 20 copies of HPV E6, E7 mRNAper cell. In all subsequent patient sample analyses, cells wereconsidered positive for HPV E6, E7 mRNA if they exhibited an MFI greaterthan 200 copies per cell, 200 representing a copy number of HPV that maynot be readily cleared from an average patient.

To validate the sensitivity and specificity of the In Cell HPV Assay atthe single cell level, the In Cell HPV Assay with flow cytometry wereused to quantitate HPV E6, E7 mRNA in a mixed cell population of HeLacells (HPV⁺) in normal ectocervical cells (HPV⁻). As shown in FIG. 2,when the serial dilutions of the mixed cells (% HPV⁺ cells) were plottedagainst the percentage of cells expressing HPV E6, E7 mRNA in the mixedcell population as determined by the In Cell HPV Assay; the result was alinear range between 0.3% and 100% for HPV E6, E7 mRNA positive cells.The signal to noise ratio in this experiment was calculated at 1.5 to2.0 logs.

Example 4 Morphological Assessment of HPV E6, E7 mRNA Message inAbnormal Cells

To confirm the specificity of the In Cell HPV Assay to detect abnormalcells, HPV⁺ HeLa cells and HPV⁻ normal ectocervical cells werehybridized with the HPV E6, E7 mRNA OncoText probes and the morphologyof cells expressing detectable levels of HPV E6, E7 mRNA message wereexamined (FIG. 3). The In Cell HPV Assay produced a characteristiccytoplasmic staining pattern in HeLa cells. By contrast, the HPV⁻ cellline did not stain (the C33A line was used) (Boxes A and B,respectively). Using the same probe cocktail used in flow cytometry,atypical cells were detected by their characteristic high nuclear tocytoplasmic ratio in a field of many normal squamous cells (Boxes C toE). An overlay image of the Nomarski phase contrast image with a 488 nMfluorescence confocal image confirmed the localization of thehybridization signal over the abnormal cells.

Example 5 Cytometric Enrichment for HPV E6, E7 Expressing Cells in aHeterogeneous Cervical Cytology Sample

Since cervical cytology specimens contain numerous cell types including,but not limited to, squamous ectocervical cells, columnar endocervicalcells, PMNs (polymorphonuclear leukocytes), and lymphocytes, an antibodycocktail was developed to be used in conjunction with the In Cell HPVAssay in order to distinguish the cell types of interest in an LBCspecimen. To distinguish between ectocervical cells, endocervical cells,and PMNs in LBC preparations of study subjects, the cervical cellspecimens were stained with a combination of CD16, a neutrophil marker,and CAM 5.2, an antibody that detects a 70 kDa molecular weightcytokeratin that is expressed in endocervical cells but not ectocervicalcells. Cells staining for a: particular marker were backgated toidentify their presence within forward and orthogonal (90° side scatter)light scatter gates to distinguish between ectocervical cells,endocervical cells, and PMNs. See, Grundhoefer and Patterson,Determination of Liquid-Based Cervical Cytology Specimen Adequacy UsingCellular Light Scatter and Flow Cytometry, CYTOMETRY 46:340-344 (2001).As shown in FIGS. 4A-4I, ectocervical cells were identified in normal(WNL) and abnormal (HGSIL) cervical cell specimens by gating for a lackof CD16 and CAM 5.2 expression (CD16⁻, CAM 5.2⁻) (FIG. 4A, first twoboxes) and ectocervical cells were analyzed for HPV E6, E7 mRNAexpression using the In Cell HPV Assay of the present invention with 100copies of HPV E6, E7 mRNA per cell used as the cut-off point fordetermining the percentage of cells in a sample expressing HPV E6, E7mRNA (FIG. 4A, third box). The percentage noted at the upper right handcorner each of FIGS. 4B-4I indicates the percentage of cells in theHGSIL (FIGS. 4B-4E) and WNL (FIGS. 4F-4I) samples that express more than100 copies of HPV mRNA per cell. In a similar vein, expression of HPVE6, E7 mRNA in endocervical cells can be analyzed by using a gatingstrategy that identifies CD16⁻, CAM 5.2⁺ expression.

Example 6 Comparative Analysis of the in Cell HPV Assay Versus the PapSmear to Detect Abnormal SILs in a High Risk Cohort

To screen for pre-malignant conditions in LBCs, LBC specimens wereobtained from a high risk cohort consisting of 41 women with normalcervical cytology and 41 women with abnormal cervical cytology. Asdescribed in Example 4, abnormal cells are identified by acharacteristically high nuclear to cytoplasmic ratio whereas normalsquamous epithelial cells exhibit a low nuclear to cytoplasmic ratio.The women with the abnormal cells consisted of the following cytologies:9 ASCUS, 22 LGSIL, and 10 HGSIL. For purposes of this experiment, thequantitative cut-off for a positive HPV E6, E7 mRNA result was set at 2standard deviations from the mean of true negative. Cervical cells werestained with CD16 and CAM 5.2 (as described in Example 5) and hybridized(as described in Example 1). Ectocervical cells were differentiated fromendocervical cells by both forward and orthogonal light scatter and byexpression of CAM 5.2 on endocervical cells but not on ectocervicalcells (Example 5, FIG. 4A). Ectocervical cells coated with neutrophilswere eliminated from analysis by the presence of CD16⁺ staining. The InCell HPV Assay detected HPV E6, E7 mRNA in 5 of 9 ASCUS samples, 13 of22 LGSIL samples, 10 of 10 HGSIL samples, and 3 of 41 normal (WNL)cervical cytology specimens. Two of the 3 normal samples had HPV E6, E7mRNA confirmed by quantitative real-time RT-PCR using Taqman probes.Similar to the procedure used in Example 3, cells were consideredpositive for HPV E6, E7 mRNA if they exhibited an MFI greater than 200copies per cell. For controls, HPV⁺ HeLa cells and HPV⁻ C33A cells wereincluded with every run. The percentage of HPV E6, E7 mRNA expressingcells positively correlated with the cytologic diagnosis as shown inTable 1.

TABLE 1 % IN CELL % IN CELL % IN CELL IN CELL HPV ASSAY CYTOLOGY HPVHIGH HPV LOW HPV MEDIAN PERFORMANCE WNL 5.6 0.1 0.5  3/41 ASCUS 7.3 0.11.9* 5/9 LGSIL 12.7 0.1 2.7** 13/22 HGSIL 46.1 2.1 13.9*** 10/10*ASCUS > WNL, p < 0.05 **LGSIL > WNL, p < 0.01; LGSIL > ASCUS, NS (notsignificant) ***HGSIL > WNL, p < 0.001; HGSIL > ASCUS, p < 0.001;HGSIL > LSIL, p < 0.001

The percentage of HPV E6, E7 mRNA expressing cells in HGSILs wassignificantly greater than the percentage of expressing cells in WNLs(p<0.001), ASCUS (p<0.001), and LGSILs p<0.001). Similarly, thepercentage of HPV E6, E7 mRNA expressing cells in LGSILs wassignificantly greater than the percentage of expressing cells in WNLs(p<0.01), but not significantly greater than the percentage of HPV E6,E7 mRNA expressing cells in ASCUS, suggesting that the molecularalterations in ASCUS more closely resemble LGSIL than HGSIL. The overallsensitivity and specificity of the In Cell HPV Assay for high gradelesions was 100% and 93%, respectively.

Example 7 Comparative Analysis of the in Cell HPV Assay with the HC IIHPV Test to Detect Abnormal SILs in a Low Risk Cohort

To compare the ability of the In Cell HPV Assay and the HC II HPV Testto detect high grade cervical lesions, 149 LBC specimens were obtainedfrom a low risk cohort with the following cytologies: 109 WNL, 21 ASCUS,5 LGSIL, 12 HGSIL, and 2 with invasive cervical cancer identified bycytologic examination. Table 2 shows the results of the comparativeanalysis. As previously discussed, the In Cell HPV Assay tests for thepresence of HPV E6, E7 mRNA while the HC II HPV Test screens for thepresence of high risk HPV DNA, e.g., HPV-16 and HPV-18.

TABLE 2 IN CELL HC II CYTOLOGY HPV ASSAY HPV TEST WNL 10/109 13/109ASCUS 14/21  9/21 LGSIL 4/5  3/5  HGSIL 8/12 8/12 Squamous Cell 2/2 1/2  Carcinoma SENSITIVITY ≧HGSIL 71.4% 64.2% SPECIFICITY ≧HGSIL   91%  88%

Similar to the results involving the high risk cohort (Example 6), thepercentage of HPV E6, E7 mRNA expressing cells detected by the In CellHPV Assay positively correlated with the cytologic diagnoses. UsingHGSIL and invasive cervical cancer as an end-point, the sensitivity andspecificity of In Cell HPV Assay to detect cervical abnormalities was71.4% and 91%, respectively, and the sensitivity and specificity of theHC II HPV Test to detect cervical abnormalities was 64.2% and 88%,respectively. Both the In Cell HPV Assay and the HC II HPV Test detected8 of 12 HGSILs, but the In Cell HPV Assay also identified an invasivecervical cancer that was missed by both real-time RT-PCR (data notshown) and the HC II HPV Test.

1. An assay for determining malignant transformation of cervical cellscomprising the steps of: (a) obtaining cervical cells from anindividual; (b) preparing the cervical cells as a liquid-based cytology(LBC) specimen; and (c) hybridizing the LBC specimen usingoligonucleotides specific for HPV E6, E7 mRNA, wherein expression of HPVE6, E7 mRNA indicates malignant transformation of the cervical cells. 2.The assay of claim 1, further comprising: (d) quantitating theexpression of HPV E6, E7 mRNA per cell via flow cytometry.
 3. The assayof claim 2, wherein expression of 2 to 1000 copies of HPV E6, E7 mRNAper cell indicates malignant transformation of the cervical cells. 4.The assay of claim 3, wherein expression of 5 to 750 copies of HPV E6,E7 mRNA per cell indicates malignant transformation of the cervicalcells.
 5. The assay of claim 4, wherein expression of 10 to 500 copiesof HPV E6, E7 mRNA per cell indicates malignant transformation of thecervical cells.
 6. The assay of claim 2, wherein expression of 200 ormore copies of HPV E6, E7 mRNA per cell indicates malignanttransformation of the cervical cells.
 7. The assay of claim 1, whereinthe cervical cells are selected from the group consisting of normalcells, abnormal squamous cells of undetermined significance (ASCUS),cells with low grade squamous intraepithelial lesions (LGSILs), andcells with high grade intraepithelial lesions (HGSILs).
 8. A method ofscreening a patient for malignant transformation of cervical cellscomprising identifying expression of HPV E6, E7 mRNA in the cervicalcells.
 9. The method of claim 8, wherein the cervical cells are selectedfrom the group consisting of normal cells, abnormal squamous cells ofundetermined significance (ASCUS), cells with low grade squamousintraepithelial lesions (LGSILs), and cells with high gradeintraepithelial lesions (HGSILs).
 10. The method of claim 8, wherein theHPV E6, E7 mRNA is identified using in situ hybridization witholigonucleotides specific for HPV E6, E7 mRNA.
 11. The method of claim10, wherein the HPV E6, E7 mRNA is quantitated using flow cytometry. 12.The method of claim 11, wherein expression of 2 to 1000 copies of HPVE6, E7 mRNA per cell indicates malignant transformation of the cervicalcells.
 13. The method of claim 10, wherein expression of 5 to 750 copiesof HPV E6, E7 mRNA per cell indicates malignant transformation of thecervical cells.
 14. The method of claim 11, wherein expression of 10 to500 copies of HPV E6, E7 mRNA per cell indicates malignanttransformation of the cervical cells.
 15. The method of claim 8, whereinexpression of 200 or more HPV E6, E7 mRNA per cells indicates malignanttransformation of the cervical cells.
 16. The method of claim 8, whereinthe HPV E6, E7 mRNA is identified using in situ hybridization witholigonucleotides specific for HPV E6, E7 mRNA.
 17. A method of screeninga patient with abnormal cervical cells for pre-malignant cervical cells,comprising quantitating the expression of HPV E6, E7 mRNA in thecervical cells, wherein cells expressing 200 or more copies of HPV E6,E7 mRNA per cell indicate cells with malignant potential and cellsexpressing less than 200 copies of HPV E6, E7 mRNA indicate benigncells.
 18. The method of claim 17, wherein the abnormal cervical cellsare selected from the group consisting of abnormal squamous cells ofundetermined significance (ASCUS), cells with low grade squamousintraepithelial lesions (LGSILs), and cells with high gradeintraepithelial lesions (HGSILs).
 19. The method of claim 17, whereinthe HPV E6, E7 mRNA is identified using in situ hybridization witholigonucleotides specific for HPV E6, E7 mRNA.
 20. The method of claim19, wherein the HPV E6, E7 mRNA is quantitated using flow cytometry. 21.An assay for identifying malignantly transformed cervical cell typescomprising the steps of: (a) obtaining cervical cells from anindividual; (b) preparing the cervical cells as a liquid-based cytology(LBC) specimen; (c) staining the cervical cells with a combination ofCD16 and CAM 5.2; and (d) hybridizing the LBC specimen usingoligonucleotides specific for HPV E6, E7 mRNA, wherein neutrophils areidentified by CD16⁺ staining, columnar endocervical cells are identifiedby CD16⁻, CAM 5.2⁺ staining, and squamous ectocervical cells areidentified by CD16⁻, CAM 5.2⁻ staining, and expression of HPV E6, E7mRNA indicates malignant transformation of the cervical cells.
 22. Theassay of claim 21, further comprising: (e) quantitating the expressionof HPV E6, E7 mRNA per cell via flow cytometry.
 23. The assay of claim22, wherein expression of 2 to 1000 copies of HPV E6, E7 mRNA per cellindicates malignant transformation of the cervical cells.
 24. The assayof claim 23, wherein expression of 5 to 750 copies of HPV E6, E7 mRNAper cell indicates malignant transformation of the cervical cells. 25.The assay of claim 24, wherein expression of 10 to 500 copies of HPV E6,E7 mRNA per cell indicates malignant transformation of the cervicalcells.
 26. The assay of claim 22, wherein expression of 200 or more HPVE6, E7 mRNA per cells indicates malignant transformation of the cervicalcells.
 27. The assay of claim 21, wherein malignantly transformedcolumnar endocervical cells indicate a pre-adenocarcinoma oradenocarcinoma cervical condition.
 28. The assay of claim 27, whereinmalignantly transformed squamous ectocervical cells indicatepre-squamous cell carcinoma or squamous cell carcinoma cervicalcondition.